Getting Started Guide

Primer Express Software Version 3.0 Getting Started Guide Before You Begin Designing Primers and Probes for Quantification Assays Designing Primers and Probes for Allelic Discrimination Assays Ordering Primers and Probes

Copyright 2004, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. Notice to Purchaser: Purchase of this software product alone does not imply any license under any process, instrument or other apparatus, system, composition, reagent or kit rights under patent claims owned or otherwise controlled by Applera Corporation, either expressly or by estoppel. TRADEMARKS: Applied Biosystems, Primer Express, ABI PRISM are registered trademarks and AB (Design), Applera, iscience, iscience (design), FAM, and TAMRA are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. TaqMan is a registered trademark of Roche Molecular Systems, Inc. SYBR is a registered trademark of Molecular Probes, Inc. Microsoft and Windows are registered trademarks of Microsoft Corporation. All other trademarks are the sole property of their respective owners. Part Number 4362460 Rev. B 1/2005

Primer Express Software Version 3.0 Workflow Chapter 1 Before You Begin Introduction Installing the Software Basics Chapter 2 Designing Primers and Probes for Quantification Assays Overview Automatically Designing Primers and Probes Manually Designing Primers and Probes Automatically Designing Primers for SYBR Green Assays Manually Designing Primers for SYBR Green Assays December 16, 2004 3:56 pm, GSG_Workflow.fm Chapter 3 Chapter 4 Designing Primers and Probes for Allelic Discrimination Assays Ordering Primers and Probes Overview Overview Automatically Designing Primers and Probes Ordering the Selected Primers and Probes Manually Designing Primers and Probes Primer Express Software Version 3.0 Getting Started Guide iii

Contents Preface vii Chapter 1 Before You Begin 1 Introduction......................................................... 2 Installing the Software................................................. 4 Basics............................................................. 6 Chapter 2 Designing Primers and Probes for Quantification Assays 7 Overview........................................................... 8 Automatically Designing Primers and Probes............................... 9 Manually Designing Primers and Probes.................................. 13 Automatically Designing Primers for SYBR Green Dye Assays............... 19 Manually Designing Primers for SYBR Green Dye Assays................... 23 December 16, 2004 3:54 pm, GSG_4tabsTOC.fm Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays 27 Overview.......................................................... 28 Automatically Designing Primers and Probes.............................. 29 Manually Designing Primers and Probes.................................. 34 Chapter 4 Ordering Primers and Probes 39 Overview.......................................................... 40 Ordering Primers and Probes.......................................... 40 Index 43 Primer Express Software Version 3.0 Getting Started Guide v

December 16, 2004 3:54 pm, GSG_4tabsTOC.fm vi Primer Express Software Version 3.0 Getting Started Guide

Preface How to Use This Guide Purpose of This Guide Audience Assumptions The Primer Express Software Version 3.0 Getting Started Guide provides instructions for automating the primer and probe design for Quantification and Allelic Discrimination assays. It also explains how to manually annotate sequences and design customized primer/prober sets. This guide is written for principal investigators and laboratory staff with general knowledge of PCR and realtime-pcr terminologies and applications. This guide assumes that you have: A working knowledge of the assays Knowledge of primer and probe definitions Familiarity with Microsoft Windows XP operating systems December 16, 2004 3:54 pm, GSG_Preface.fm Text Conventions Bold indicates user action. For example: Type 0, then press Enter for each of the remaining fields. Italic text indicates new or important words and is also used for emphasis. For example: Before analyzing, always prepare fresh matrix. A right arrow bracket (>) separates successive commands you select from a dropdown or shortcut menu. For example: Select File > Open > Spot Set. User Attention Words Two user attention words appear in Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below: Note: Provides information that may be of interest or help but is not critical to the use of the product. IMPORTANT! Provides information that is necessary for proper instrument operation, accurate chemistry kit use, safe use of a chemical, or proper software use. Primer Express Version 3.0 Getting Started Guide vii

Preface How to Obtain More Information Examples of the user attention words appear below: Note: The size of the column affects the run time. IMPORTANT! To verify your client connection to the database, you need a valid Oracle user ID and password. How to Obtain More Information For more information about using Primer Express Software, refer to the comprehensive on line help system, which includes context-sensitive help and detailed procedures for performing tasks. The help system can be invoked by pressing the F1 key anywhere in the software. Send Us Your Comments Applied Biosystems welcomes your comments and suggestions for improving its user documents. You can e-mail your comments to: techpubs@appliedbiosystems.com How to Obtain Support For the latest services and support information for all locations, go to http://www.appliedbiosystems.com, then click the link for Support. At the Support page, you can: Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities. Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents Search through frequently asked questions (FAQs) Submit a question directly to Technical Support Download PDF documents Obtain information about customer training Download software updates and patches December 16, 2004 3:54 pm, GSG_Preface.fm viii Primer Express Version 3.0 Getting Started Guide

Chapter 1 Before You Begin 1 Before You Begin Introduction See page 2 Installing the Software See page 4 Designing Primers and Probes for Quantification Assays Basics See page 6 December 16, 2004 3:54 pm, GSG_Chapter_4-01.fm Designing Primers and Probes for Allelic Discrimination Assays Ordering Primers and Probes See page 2 See page 2 See page 2 Primer Express Software 3.0 Getting Started Guide 1

Chapter 1 Before You Begin Introduction Introduction About Primer Express Software Primer Express software is a primer and probe design tool made specifically for use with the following instruments: Applied Biosystems 7900HT Fast Real-Time PCR System Applied Biosystems 7500 Fast Real-Time PCR System Applied Biosystems 7500 Real-Time PCR System Applied Biosystems 7300 Real-Time PCR System Primer Express software lets you independently design oligonucleotides (oligos) for PCR applications using a customized application specific document for each of the following assay types: Absolute/Relative Quantification Allelic Discrimination When using Primer Express software, keep in mind the Applied Biosystems Rapid Assay Development Guidelines that contain the following important components: Design of primers and probes using Primer Express software Selection of the appropriate reagent configuration (TaqMan Universal PCR Master Mix or SYBR Green PCR Master Mix) Use of universal thermal cycling parameters Use of default primer and probe concentrations (or optimizing, if necessary) IMPORTANT! These components provide a rapid and reliable system for assay design and optimization only when used in their entirety. Due to the interdependence of many of the individual components, the system must be adopted as a whole in order to achieve the highest level of success. Terms You Need to Know Allelic Discrimination Assay An assay that discriminates between two alleles of single nucleotide polymorphisms (SNPs). TaqMan allelic discrimination assays use two probes specific for the two possible SNP variants. Anti-Sense Strand In double-stranded DNA, the strand that does not code for the RNA, and is not translated into proteins. Also referred to as anti-coding, negative, or reverse strand. The Primer Express Software designs primers and probes using the sense strand, not the anti-sense strand. Document In the Primer Express Software, a container used to hold sequences, generate candidate primer and probe designs, and order candidate primer and probe designs. The four document types available are TaqMan MGB Quantification, TaqMan Quantification, TaqMan MGB Allelic Discrimination, and TaqMan Allelic Discrimination. December 16, 2004 3:54 pm, GSG_Chapter_4-01.fm 2 Primer Express Software 3.0 Getting Started Guide

Chapter 1 Before You Begin Introduction Primer A complementary oligonucleotide that initiates amplification of a target region of DNA. A forward primer anneals to the anti-sense strand. A reverse primer anneals to the sense strand. 1 Probe A short oligonucleotide sequence that anneals specifically to a target sequence and serves as a fluorescence monitoring system for DNA amplification. TaqMan MGB Probe An oligonucleotide with a reporter fluorescent dye attached to the 5 end and a non-fluorescent quencher attached to the 3 end. The probe is coupled with a minor groove binder (MGB), which increases its Tm. When the probe is cleaved by the DNA polymerase during the PCR reaction, reporter dye fluorescence increases proportional to the quantity of the target sequence. TaqMan Probe An oligonucleotide with a reporter fluorescent dye attached to the 5 end and a quencher fluorescent dye (usually TAMRA ) attached to the 3 end. When the probe is cleaved by the DNA polymerase during the PCR reaction, reporter dye fluorescence increases proportional to the quantity of the target sequence. Quantification Assay An assay that determines the relative or absolute quantity of target sequence within a sample. Relative quantification measures the change in the expression of the target gene in a test sample, relative to a calibrator sample. Absolute quantification uses a standard curve to calculate the quantity of an unknown target sequence. Sense Strand In double-stranded DNA, the strand that codes for the RNA that is translated into proteins. Also referred to as coding, forward, or positive strand. The Primer Express Software designs primers and probes using the sense strand. System Requirements The following table lists the hardware and software requirements and recommendations for installing and using Primer Express version 3.0 software. Item Minimum Requirements Recommendations December 16, 2004 3:54 pm, GSG_Chapter_4-01.fm Computer Intel Pentium III processor 540 MHz Monitor 17-inch monitor 800 x 600 pixels resolution Hard Drives 256 MB RAM 20 MB free hard disk space Network Adaptors 10/100 NIC with RWV (internal) Printer Any PC-compatible printer. Operating System Windows XP Professional, Service Pack 1 or later Operating Systems Not Supported Microsoft Windows NT and 2000 Macintosh Intel Pentium IV processor faster than 2GHz 19-inch or larger monitor 1024 x 768 pixels or higher pixels resolution 512 MB RAM 10 GB EIDE hard drive Windows XP Professional, Service Pack 1 or later Primer Express Software 3.0 Getting Started Guide 3

Chapter 1 Before You Begin Installing the Software Installing the Software Installing Primer Express Software Version 3.0 Note: Applied Biosystems recommends that you disable any virus protection software enabled on your computer before installing Primer Express software version 3.0. You can enable the virus protection software after installation is complete. 1. Insert the Primer Express 3.0 software CD into your CD drive. 2. If the Primer Express Installer does not start automatically, in Windows Explorer, locate and then double-click the Setup.exe file. The Primer Express software displays the following window: To Uninstall Primer Express Software Version 3.0 3. Click Install Primer Express and follow the prompts to complete the installation. IMPORTANT! Do not over write Primer Express Software version 2.0. The default installation location for Primer Express Software version 3.0 is in the Windows Start > All Programs > Applied Biosystems menu. If you change this default, verify that you are not installing it in the version 2.0 folder. You will need Primer Express Software version 2.0 to convert any older files. See Converting Primer Express Software Version 2.0 Documents on page 5. You can start using Primer Express software without restarting your computer. To uninstall Primer Express Software: 1. On the taskbar, select Start > Control Panel. The Control Panel window opens. December 16, 2004 3:54 pm, GSG_Chapter_4-01.fm 4 Primer Express Software 3.0 Getting Started Guide

December 16, 2004 3:54 pm, GSG_Chapter_4-01.fm Chapter 1 Before You Begin Installing the Software 2. In the Name column, double-click Add or Remove Programs.The Add or Remove Programs window opens. 3. In the Currently installed programs box, scroll down to, and then click Primer Express 3.0. 1 4. Click Change/Remove. 5. Follow the instructions on the Install Shield Wizard to remove all installed features. Converting Primer Express Software Version 2.0 Documents In Primer Express software version 2.0, information about your oligonucleotide designs was stored in one or more archive files. In Primer Express software version 3.0, information about each oligonucleotide design is saved to its own separate *.pxd file. If you would like to use Primer Express version 2.0 documents in version 3.0, you must first convert the version 2.0 archive files. Use the Primer Express version 2.0 Export command to convert documents within *.pcr files to individual *.pex documents. For more information, see Primer Express Software v2.0 User s Manual (PN 4329500). You can open version 2.0 *.pex files without converting to *.pxd. Note: If the Limit 3 G+C checkbox was unchecked in documents created in Primer Express Software version 2.0, this parameter (in the converted file) will be inconsistent with the setting from the version 2.0 document. Re-run the design with the Max Primer 3 GCs set to -1 to obtain consistent results. Primer Express Software 3.0 Getting Started Guide 5

Chapter 1 Before You Begin Basics Basics Starting and Exiting Primer Express Software To start Primer Express software for the first time: 1. On your desktop, select Start > Programs > Applied Biosystems > Primer Express > Primer Express 3.0. After you start Primer Express 3.0 for the first time, the registration dialog box opens. 2. Enter your name, your organization, and your registration code, which is located on your Primer Express CD envelope and paper. IMPORTANT! Be sure to store your Primer Express software registration code in a safe place. You will need it after the first installation and any re-installation. If it is lost, you must repurchase Primer Express Software. 3. Click OK. To exit Primer Express software: Select File > Exit. Using Online Help The Primer Express Software Online Help provides context-sensitive help for most windows in the software. It also provides more general information about the software and procedures for common tasks. Press F1 on the keyboard to display information about the window or dialog box you are viewing. Select Help > Contents and Index to display the default help topic. Refer to Primer Express Software Version 3.0 Online Help for more information on these Primer Express software functions: Annotating Sequences Exporting Printing Using the Batch Process Tool December 16, 2004 3:54 pm, GSG_Chapter_4-01.fm 6 Primer Express Software 3.0 Getting Started Guide

Chapter 2 Designing Primers and Probes for Quantification Assays Before You Begin 2 Designing Primers and Probes for Quantification Assays Overview See page 8 Automatically Designing Primers and Probes Seepage9 December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm Designing Primers and Probes for Allelic Discrimination Assays Ordering Primers and Probes Manually Designing Primers and Probes Automatically Designing Primers for SYBR Green Assays Manually Designing Primers for SYBR Green Assays Seepage13 Seepage19 Seepage23 Primer Express Software 3.0 Getting Started Guide 7

Chapter 2 Designing Primers and Probes for Quantification Assays Overview Overview About This Chapter Workflow This chapter provides information on using Primer Express Software Version 3.0 to automatically design primers and probes for quantification assays (including SYBR Green Dye Assays) using default parameters. It also includes information on how to manually design primers and probes to obtain customized results. Quantification Workflow Open or Create a Quantification Document Load a DNA Sequence File Find Primers and Probes View Results No Primers and Probes Found? Yes No Edit Default Parameters Figure 1. Quantification workflow Manually Design the Probe Manually Design the Primers Desired Primers and Probes Found? Yes Order Primers and Probes Order Primers and Probes December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 8 Primer Express Software 3.0 Getting Started Guide

Chapter 2 Designing Primers and Probes for Quantification Assays Automatically Designing Primers and Probes Automatically Designing Primers and Probes This section describes automatically designing primers and probes for one sequence. You can automatically design quantification primers and probes for multiple sequences using the Batch Process Tool. For more information, see Primer Express Software Version 3.0 Online Help. Creating a Quantification Document To create a new quantification document: 1. Select File > New. The New dialog box opens. Loading a DNA Sequence File 2. In the Type list, select TaqMan MGB Quantification or TaqMan Quantification. 3. Click OK. The document window opens to the Sequence tab. A sample sequence, NM_002217, is located in the sample sequences folder within the Primer Express folder. You can use this sample file to experiment with the software and design your primers and probes. To load a sequence file: 1. Select Tools > Add DNA File ( ). Note you can also copy and paste or type your sequence file in the Sequence tab. 2. At the Add DNA File dialog box, navigate to and select the desired file. For information on the various file formats supported, see Primer Express Software Version 3.0 Online Help. 2 December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 3. Click Add. Primer Express software loads the nucleotide sequence from the file and displays the sense strand in the Sequence tab (see Figure 2. Sequence tab on page 10). The sequence serves as the starting point for primer and probe design. Primer Express Software 3.0 Getting Started Guide 9

Chapter 2 Designing Primers and Probes for Quantification Assays Automatically Designing Primers and Probes Figure 2. Sequence tab Note: If you select the Double-Stranded checkbox in the Sequence tab, both sense and anti-sense strands will be displayed. However, primers and probes are designed using the sense strand sequence only. Finding Primers and Probes To find primers and probes: Select Tools > Find Primers/Probes ( ). Primer Express software performs its calculations based on default parameter values. The status bar, located at the bottom of the window, displays information about the progress of the calculations as the software searches for primer/probe sets. If primers and probes are found, go to Viewing Results on page 11. If primers and probes were not found: If the software does not find primers and probes using default parameters, a pop-up will appear stating that no acceptable primer pairs were found and that you can see the Interim Results window. For more information on Interim Results, see Primer Express Software Version 3.0 Online Help. December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 10 Primer Express Software 3.0 Getting Started Guide

Chapter 2 Designing Primers and Probes for Quantification Assays Automatically Designing Primers and Probes At this point, you can: Manually design primers and probes as described in Manually Designing Primers and Probes on page 13. Design using the complementary sequence. For more information, see Primer Express Software Version 3.0 Online Help. Viewing Results Primer Express software automatically displays the Primers/Probes tab, if it finds primers and probes. The Primers/Probes tab displays the Candidate Primers & Probes table that contains information about the candidate primers, probes, and amplicons (see Figure 3. Primers/Probes tab displaying candidate primers and probes ). The forward primer sequences are displayed using the left-to-right 5 -to-3 convention, and reverse primer sequences are displayed using the right-to-left 5 -to-3 convention. 2 December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm Figure 3. Primers/Probes tab displaying candidate primers and probes Primer Express Software 3.0 Getting Started Guide 11

Chapter 2 Designing Primers and Probes for Quantification Assays Automatically Designing Primers and Probes Evaluating the candidate primer and probe sets: The Location section of the Primers/Probes tab illustrates the location of the primers and probes within the sequence. The number above the line is the starting base; the number below the line is the ending base. Note that you can also see the corresponding location of a selected candidate Primer/Probe set in the Sequence tab. In the Sequence tab, the probe will be highlighted in pink, the forward primer in blue, and the reverse primer in yellow. These default color designations can be changed by clicking Tools > Options. If you place your cursor over any of these annotations, a tool tip will appear showing the name of the annotation (Probe, Forward Primer, Reverse Primers) start and end locations, Tm and %GC. As a general guideline, select the primer/probe sets with a low Penalty score and a low amplicon length (if the Penalty score and Amplicon Length fields are not displayed, scroll to the right in the table). However, all primer/probe sets generated using default parameters meet primer and probe guidelines. For more information regarding Penalty scores, see Primer Express Software Version 3.0 Online Help. Note: After the software finds primers and probes, the sequence box is locked. To edit the sequence, click to unlock. Saving the Document Ordering Primers and Probes Select File > Save As to save the document for future use. To order your selected primers and probes, refer to Chapter 4, Ordering Primers and Probes. IMPORTANT! Before running your samples, Applied Biosystems recommends that you run control samples to verify the performance of the selected primers and probes. December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 12 Primer Express Software 3.0 Getting Started Guide

Chapter 2 Designing Primers and Probes for Quantification Assays Manually Designing Primers and Probes Manually Designing Primers and Probes You may choose to manually design primers and probes for various reasons: Automated primer/probe design did not find primers and probes. To design a probe over an exon junction. To design a probe for DNA sequence homologs. To design primers and probes according to your own specifications. Creating a Quantification Document Manually Designing the Probe Create a Quantification document and load a sequence file as you would for automatic primer/probe set design. See Creating a Quantification Document on page 9. 1. Select a putative probe region containing at least 25 bases. 2. Copy (Ctrl+C) the sequence. IMPORTANT! The Primer Probe Test Tool eliminates non-atcg bases. Before copying a sequence, change any non-atcg bases, or select a different region of the sequence. 2 3. Select Tools > Primer Probe Test Tool. The Primer Probe Test Tool dialog box appears (see Figure 4. Primer Probe Test Tool dialog box on page 14). December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm Primer Express Software 3.0 Getting Started Guide 13

Chapter 2 Designing Primers and Probes for Quantification Assays Manually Designing Primers and Probes Figure 4. Primer Probe Test Tool dialog box 4. From the Document Type drop down menu, select the desired document type. Verify that the Parameter box is set to Default. For more information about changing parameters, see Primer Express Software Version 3.0 Online Help. 5. Paste (Ctrl+V) the putative sequence in the Probe 1 field. The Primer Probe Test Tool displays the Tm, %GC, and the oligonucleotide length to the right of the Probe 1 field. 6. If the Tm is not between 68 C to 70 C, highlight a section of the sequence to view the corresponding Tm, %GC, and oligonucleotide length. Once the highlighted region results in the desired Tm, click on Trim to delete the non-highlighted bases. Ensure the following guidelines are met (for more information on design guidelines, refer to Primer Express Software Version 3.0 Online Help): Amplicon Length 50 to 150 bases for optimum PCR efficiency. Probe Length 13 to 25 bases (13 to 30 bases if using conventional TaqMan probes) Tm 68 C to 70 C. % GC 30% to 80%. December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 14 Primer Express Software 3.0 Getting Started Guide

Chapter 2 Designing Primers and Probes for Quantification Assays Manually Designing Primers and Probes 5 end Cannot be a G residue. A G residue adjacent to the reporter dye will quench the reporter fluorescence somewhat, even after cleavage. Avoid the following motifs: Repeating oligonucleotides Avoid runs of identical nucleotides. If repeats are present, there must be fewer than four consecutive G residues. Consecutive A residues Avoid six consecutive A residues anywhere in the probe. G residues on the 3 end Avoid 5 -...GGG-MGB-3 or 5 -...GGAG-MGB-3 CC dinucleotides Avoid two or more CC dinucleotides in the middle of the probe (TaqMan MGB probes), which can sometimes reduce signal. FAM dye-labeled probes If ordering FAM -dye labeled probes, avoid a G in the second position on the 5 end. For secondary structure design considerations, see Primer Express Software Version 3.0 Online Help. Note: If you cannot achieve the recommended Tm, you can design using the complementary sequence. For more information, see Primer Express Software Version 3.0 Online Help. 2 7. Once the correct Tm is achieved, return to the Sequence tab and highlight the sequence found in the Probe 1 field of the Primer Probe Test Tool. To manually design primers, go to Manually Designing the Primers on page 16. December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 8. To automatically find primers after manually designing the probe, select Edit > Annotate > Probe ( ). The selected probe sequence text is displayed in green. For more information on annotating sequences, see Primer Express Software Version 3.0 Online Help. 9. Select Tools > Find Primers/Probes ( ). Primer Express software performs its calculations based on default parameter values. The status bar, located at the bottom of the window, displays information about the progress of the calculations as the software searches for primers based on the designed probe. If primers are found, go to Viewing Results on page 16. If primers were not found: If the software does not find primers using default parameters, a pop-up will appear stating that no acceptable primer pairs were found and that you can see the Interim Results window. For more information on Interim Results, see Primer Express Software Version 3.0 Online Help. At this point, you can manually design primers described in Manually Designing the Primers on page 16. Primer Express Software 3.0 Getting Started Guide 15

Chapter 2 Designing Primers and Probes for Quantification Assays Manually Designing Primers and Probes Viewing Results Primer Express software automatically displays the Primers/Probes tab, if it finds primers. The Primers/Probes tab displays the Candidate Primers & Probes table that contains information about the candidate primers, probes, and amplicons. The forward primer sequences are displayed using the left-to-right 5 -to-3 convention, and reverse primer sequences are displayed using the right-to-left 5 -to-3 convention. Evaluating the candidate primer and probe sets: The Location section of the Primers/Probes tab illustrates the location of the primers and probes within the sequence. The number above the line is the starting base; the number below the line is the ending base. Note that you can also see the corresponding location of a selected candidate Primer/Probe set in the Sequence tab. In the Sequence tab, the probe will be highlighted in pink, the forward primer in blue, and the reverse primer in yellow. These default color designations can be changed by clicking Tools > Options. If you place your cursor over any of these annotations, a tool tip will appear showing the name of the annotation (Probe, Forward Primer, Reverse Primers) start and end locations, Tm and %GC. As a general guideline, select the primer/probe sets with a low Penalty score and a low amplicon length (if the Penalty score and Amplicon Length fields are not displayed, scroll to the right in the table). However, all primer/probe sets generated using default parameters meet primer and probe guidelines. For more information regarding Penalty scores, see Primer Express Software Version 3.0 Online Help. Note: After the software finds primers and probes, the sequence box is locked. To edit the sequence, click to unlock. Manually Designing the Primers To design the Forward Primer: 1. Select a sequence (at least 25 bases) to the left of the probe. The sequence should be as close to the probe as possible without overlapping it. 2. Copy (Ctrl+C) the sequence. IMPORTANT! The Primer Probe Test Tool eliminates non-atcg bases. Before copying a sequence, change any non-atcg bases, or select a different region of the sequence. 3. On the Primer Probe Test Tool dialog box, paste (Ctrl+V) the sequence into the Fwd Primer field. The Primer Probe Test Tool displays the Tm, %GC, and the oligonucleotide length to the right of the Fwd Primer field. December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 16 Primer Express Software 3.0 Getting Started Guide

Chapter 2 Designing Primers and Probes for Quantification Assays Manually Designing Primers and Probes 4. If the Tm is not between 58 C to 60 C, highlight a section of the sequence to view the corresponding Tm, %GC, and oligonucleotide length as if those highlighted bases were deleted. Once the highlighted region results in the desired Tm, click on Trim to delete the non-highlighted bases. Ensure the following guidelines are met (for more information on design guidelines, refer to Primer Express Software Version 3.0 Online Help): Amplicon Length 50 to 150 bases for optimum PCR efficiency. Optimal Primer Length 20 bases. Do not overlap primer and probe sequences. Tm 58 C to 60 C (Optimal Tm 59 C). % GC 30% to 80%. 3 end Make sure the last five nucleotides at the 3 end contain no more than two G + C residues. Avoid the following motifs: Repeating oligonucleotides Avoid runs of identical nucleotides. If repeats are present, there must be fewer than four consecutive G residues. For secondary structure design considerations, see Primer Express Software Version 3.0 Online Help. 2 To design the Reverse Primer: 1. In the sequence tab, select a sequence (at least 25 bases) to the right of the probe. The sequence should be as close to the probe without overlapping it. 2. Select Edit > Copy Complement. December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm Saving Primer and Probe Sequences IMPORTANT! The Primer Probe Test Tool eliminates non-atcg bases. Before copying a sequence, change any non-atcg bases, or select a different region of the sequence. 3. On the Primer Probe Test Tool dialog box, paste (Ctrl+V) the sequence into the Rev Primer field. The Primer Probe Test Tool displays the Tm, %GC, and the oligonucleotide length to the right of the Fwd Primer field. 4. If the Tm is not between 58 C to 60 C, highlight a section of the sequence to view the corresponding Tm, %GC, and oligonucleotide length. Once the highlighted region results in the desired Tm, click on Trim to delete the non-highlighted bases. Be sure to keep the above guidelines in mind. Note that you can further customize your primer and probe set by editing the default parameter values found under the Parameters tab. For more information on editing parameters, see Primer Express 3.0 Software Online Help. Copy and paste the primer and probe sequences into a text document, then save for future reference. Primer Express Software 3.0 Getting Started Guide 17

Chapter 2 Designing Primers and Probes for Quantification Assays Manually Designing Primers and Probes Ordering Primers and Probes To order your selected primer/probe set, refer to Chapter 4, Ordering Primers and Probes. IMPORTANT! Before running your samples, Applied Biosystems recommends that you run control samples to verify the performance of the selected primers and probes. December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 18 Primer Express Software 3.0 Getting Started Guide

Chapter 2 Designing Primers and Probes for Quantification Assays Automatically Designing Primers for SYBR Green Dye Assays Automatically Designing Primers for SYBR Green Dye Assays Note: This procedure generates primers and TaqMan probes. However, only the primers need to be ordered for SYBR Green Dye assays. If desired, you can save the probe sequence for future use in TaqMan assays. Creating a Quantification Document To create a new quantification document: 1. Select File > New. The New dialog box opens. 2. In the Type list, select TaqMan MGB Quantification or TaqMan Quantification. 3. Click OK. The document window opens to the Sequence tab. 2 Loading a DNA Sequence File A sample sequence NM_002217, is located in the sample sequences folder within the Primer Express folder. You can use this sample file to experiment with the software and design your primers. To load a sequence file: 1. Select Tools > Add DNA File ( ). Note you can also copy and paste or type your sequence file in the Sequence tab. December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 2. At the Add DNA File dialog box, navigate to and select the desired file. For information on the various file formats supported, see Primer Express Software Version 3.0 Online Help. 3. Click Add. Primer Express software loads the nucleotide sequence from the file and displays the sense strand in the Sequence tab (see Figure 5. Sequence tab on page 20). The sequence serves as the starting point for primer design. Primer Express Software 3.0 Getting Started Guide 19

Chapter 2 Designing Primers and Probes for Quantification Assays Automatically Designing Primers for SYBR Green Dye Assays Figure 5. Sequence tab Note: If you select the Double-Stranded checkbox in the Sequence tab, both sense and reverse strands will be displayed. However, primers are designed using the sense strand sequence only. Finding Primers To find primers: Select Tools > Find Primers/Probes ( ). Primer Express software performs its calculations based on default parameter values. The status bar, located at the bottom of the window, displays information about the progress of the calculations as the software searches for primer/probe sets. If primers are found, go to Viewing Results on page 21. If primers were not found: If the software does not find primers using default parameters, a pop-up will appear stating that no acceptable primer pairs were found and that you can see the Interim Results window. For more information on Interim Results, see Primer Express Software Version 3.0 Online Help. December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 20 Primer Express Software 3.0 Getting Started Guide

Chapter 2 Designing Primers and Probes for Quantification Assays Automatically Designing Primers for SYBR Green Dye Assays At this point, you can: Manually design primers as described in Manually Designing Primers for SYBR Green Dye Assays on page 23. Design using the complementary sequence. For more information, see Primer Express Software Version 3.0 Online Help. Viewing Results Primer Express software automatically displays the Primers/Probes tab, if it finds primers and probes. The Primers/Probes tab displays the Candidate Primers & Probes table that contains information about the candidate primers, probes, and amplicons (see Figure 6. Primers/Probes tab displaying candidate primers and probes ). The forward primer sequences are displayed using the left-to-right 5 -to-3 convention, and reverse primer sequence using the right-to-left 5 -to-3 convention. 2 December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm Figure 6. Primers/Probes tab displaying candidate primers and probes Primer Express Software 3.0 Getting Started Guide 21

Chapter 2 Designing Primers and Probes for Quantification Assays Automatically Designing Primers for SYBR Green Dye Assays Evaluating the candidate primer and probe sets: The Location section of the Primers/Probes tab illustrates the location of the primers and probes within the sequence. The number above the line is the starting base; the number below the line is the ending base. Note that you can also see the corresponding location of a selected candidate Primer/Probe set in the Sequence tab. In the Sequence tab, the probe will be highlighted in pink, the forward primer in blue, and the reverse primer in yellow. These default color designations can be changed by clicking Tools > Options. If you place your cursor over any of these annotations, a tool tip will appear showing the name of the annotation (Probe, Forward Primer, Reverse Primers) start and end locations, Tm and %GC. As a general guideline, select the primer/probe sets with a low Penalty score and a low amplicon length (if the Penalty score and Amplicon Length fields are not displayed, scroll to the right in the table). However, all primer/probe sets generated using default parameters meet primer and probe guidelines. For more information regarding Penalty scores, see Primer Express Software Version 3.0 Online Help. Note: After the software finds primers and probes, the sequence box is locked. To edit the sequence, click to unlock. Saving the Document Ordering Primers Select File > Save As to save the document for future use. To order your selected primers, refer to Chapter 4, Ordering Primers and Probes. IMPORTANT! Before running your samples, Applied Biosystems recommends that you run control samples to verify the performance of the selected primers and probes. December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 22 Primer Express Software 3.0 Getting Started Guide

Chapter 2 Designing Primers and Probes for Quantification Assays Manually Designing Primers for SYBR Green Dye Assays Manually Designing Primers for SYBR Green Dye Assays You may choose to manually design primers and probes for a various reasons: Automated primer/probe design did not find primers. To design primers according to your own specifications. Creating a Quantification Document Manually Designing the Primers Create a Quantification document and load a sequence file as you would for automatic primer/probe set design. See Creating a Quantification Document on page 9. To design the Forward Primer: 1. In the Sequence tab, select a putative forward primer sequence region containing at least 25 bases. 2. Copy (Ctrl+C) the sequence. 2 IMPORTANT! The Primer Probe Test Tool eliminates non-atcg bases. Before copying a sequence, change any non-atcg bases, or select a different region of the sequence. 3. Select Tools > Primer Probe Test Tool. The Primer Probe Test Tool dialog box appears (see Figure 7. Primer Probe Test Tool dialog box on page 24). December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm Primer Express Software 3.0 Getting Started Guide 23

Chapter 2 Designing Primers and Probes for Quantification Assays Manually Designing Primers for SYBR Green Dye Assays Figure 7. Primer Probe Test Tool dialog box 4. From the Document Type drop down menu, select the desired document type. Verify that the Parameter field is set to Default. For more information about changing parameters, see Primer Express Software Version 3.0 Online Help. 5. Paste (Ctrl+V) the annotated sequence in the Fwd Primer field. The software displays the Tm, %GC, and the oligonucleotide length to the right of the Fwd Primer field. 6. If the Tm is not between 58 C to 60 C, highlight a section of the sequence to view the corresponding Tm, %GC, and oligonucleotide length as if those highlighted bases were deleted. Once the highlighted region results in the desired Tm, click on Trim to delete the highlighted bases. Ensure the following guidelines are met (for more information on design guidelines, refer to Primer Express Software Online Help): Amplicon Length 50 to 150 bases for optimum PCR efficiency. Optimal Primer Length 20 bases. Do not overlap primer and probe sequences. Tm 58 C to 60 C (Optimal Tm 59 C). % GC 30% to 80%. December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 24 Primer Express Software 3.0 Getting Started Guide

Chapter 2 Designing Primers and Probes for Quantification Assays Manually Designing Primers for SYBR Green Dye Assays 3 end Make sure the last five nucleotides at the 3 end contain no more than two G + C residues. Avoid the following motifs: Repeating oligonucleotides Avoid runs of identical nucleotides. If repeats are present, there must be fewer than four consecutive G residues. For secondary structure design considerations, see Primer Express Software Version 3.0 Online Help. Note: If you cannot achieve the recommended Tm, you can design using the complementary sequence. For more information, see Primer Express Software Version 3.0 Online Help. To design the Reverse Primer: 1. In the sequence tab, select a putative reverse primer sequence region (containing at least 25 bases). 2 IMPORTANT! The Primer Probe Test Tool eliminates non-atcg bases. Before copying a sequence, change any non-atcg bases, or select a different region of the sequence. 2. Select Edit > Copy Complement. 3. On the Primer Probe Test Tool dialog box, paste (Ctrl+V) the primer sequence into the Rev Primer field. The Primer Probe Test Tool displays the Tm, %GC, and the oligonucleotide length to the right of the Rev Primer field. December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm Saving Primer Sequences Ordering Primers 4. If the Tm is not between 58 C to 60 C, highlight a section of the sequence to view the corresponding Tm, %GC, and oligonucleotide length. Once the highlighted region results in the desired Tm, click on Trim to delete the non-highlighted bases. Be sure to keep the above guidelines in mind. Note that you can further customize your primer by editing the default parameter values found under the Parameters tab. For more information on editing parameters, see Primer Express 3.0 Software Online Help. Copy and paste the primer and probe sequences into a text document, then save for future reference. To order your selected primer/probe set, refer to Chapter 4, Ordering Primers and Probes. IMPORTANT! Before running your samples, Applied Biosystems recommends that you run control samples to verify the performance of the selected primers and probes. Primer Express Software 3.0 Getting Started Guide 25

Chapter 2 Designing Primers and Probes for Quantification Assays Manually Designing Primers for SYBR Green Dye Assays December 16, 2004 3:54 pm, GSG_Chapter_4-02.fm 26 Primer Express Software 3.0 Getting Started Guide

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays Before You Begin Designing Primers and Probes for Quantification Assays December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm Designing Primers and Probes for Allelic Discrimination Asays Ordering Primers and Probes Overview Automatically Designing Primers and Probes Manually Designing Primers and Probes Seepage28 See page 29 See page 34 3 Primer Express Software 3.0 Getting Started Guide 27

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays Overview Overview About This Chapter Workflow This chapter provides information on using Primer Express software to automatically design primers and probes for allelic discrimination assays using default parameters. It also includes information on how to manually design primers and probes to obtain customized results. Allelic Discrimination Workflow Open or Create an Allelic Discrimination Document Load a DNA Sequence File Assign a SNP Target Find Primers and Probes View Results No Primers and Probes Found? Yes No Edit Default Parameters Manually Design Probe 1 Manually Design Probe 2 Manually Design the Primers Desired Primers and Probes Found? Figure 8. Allelic Discrimination workflow Yes Order Primers and Probes Order Primers and Probes December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm 28 Primer Express Software 3.0 Getting Started Guide

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays Automatically Designing Primers and Probes Automatically Designing Primers and Probes Creating an Allelic Discrimination Document To create a new allelic discrimination document: 1. Select File > New to open the New dialog box. 2. In the Type list, select TaqMan MGB Allelic Discrimination or TaqMan Allelic Discrimination. For best results, use TaqMan MGB probes. MGB probes are shorter than conventional probes and are more specific to the target sequence. 3. Click OK. Loading a DNA Sequence File A sample sequence file, AY228765.txt, is located in the sample sequences folder within the Primer Express folder. You can use this sample file to experiment with the software and design your primers and probes. Note that one forward primer, one reverse primer, and two probes are designed. The two probes, one for each of the SNP sites, will not be identical. However, the two probes must be designed using the same strand (sense strand). To load a sequence file: 1. Select Tools > Add DNA File ( ). Note you can also copy and paste or type your sequence file in the Sequence tab. 2. At the Add DNA File dialog box, navigate to and select the desired file. For information on the various file formats supported, see Primer Express Software Version 3.0 Online Help. December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm 3. Click Add. Primer Express software loads the nucleotide sequence from the file and displays the sense strand in the Sequence tab (see Figure 9. Sequence tab on page 30). The sequence serves as the starting point for primer and probe design. 3 Primer Express Software 3.0 Getting Started Guide 29

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays Automatically Designing Primers and Probes Figure 9. Sequence tab Note: If you select the Double-Stranded checkbox in the Sequence tab, both sense and anti-sense strands will be displayed. However, primers and probes are designed using the sense strand sequence only. Assigning a SNP Target To assign a SNP target: 1. Highlight the SNP target site. 2. Select Edit > Annotate > SNP Target ( ) then select the variant for the SNP site. To determine the variant to select, find the two possible variant bases for your SNP, then click the code between the two bases. In the example sequence provided, the SNP target is located at position 528 as a G/A variant, so click R, then OK (see Figure 10. Determining variant using SNP Target Tool on page 31). December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm 30 Primer Express Software 3.0 Getting Started Guide

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays Automatically Designing Primers and Probes Figure 10. Determining variant using SNP Target Tool Finding Primers and Probes To find primers and probes: Select Tools > Find Primers/Probes ( ). Primer Express software performs its calculations based on default parameter values. December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm Viewing Results The status bar, located at the bottom of the window, displays information about the progress of the calculations as the software searches for primer, probe, and amplicon sets. If primers and probes are found, go to Viewing Results. If primers and probes were not found: If the software does not find primers and probes using default parameters, a pop-up will appear stating that no acceptable primer pairs were found and that you can see the Interim Results window. For more information on Interim Results, see Primer Express Software Version 3.0 Online Help. Primer Express software automatically displays the Primers/Probes tabs if it finds primers and probes (see Figure 11. Primer/Probe Tab displaying candidate primers and probes on page 32). The Primers/Probes tab displays the candidate Primers & Probes table that contains information about forward primers, reverse primers, probes, and amplicons. The forward primer sequences are displayed using the left-to-right 5 -to-3 convention, and reverse primer sequences are displayed using the right-to-left 5 -to-3 convention. 3 Primer Express Software 3.0 Getting Started Guide 31

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays Automatically Designing Primers and Probes Figure 11. Primer/Probe Tab displaying candidate primers and probes Evaluating the candidate primer and probe sets: The Location section illustrates the location of the primers and probes within the line sequence. The number above the line is the starting base; the number below the line is the ending base. Note that you can also see the corresponding location of a selected candidate Primer/Probe set in the Sequence tab. In the sequence tab, the probe 1 will be highlighted in pink, probe 2 will be highlighted in green (if probe 1 and 2 overlap, the overlap region will appear green), the forward primer in blue, and the reverse primer in yellow (see Figure 12. Probe annotations in Sequence tab on page 33). These default color designations can be changed by clicking Tools > Options. If you place your cursor over any of these annotations, a tool tip will appear showing the name of the annotation (Probe, Forward Primer, Reverse Primers) start and end locations, Tm and GC%. December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm 32 Primer Express Software 3.0 Getting Started Guide

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays Automatically Designing Primers and Probes Figure 12. Probe annotations in Sequence tab Note: After the software finds primers and probes, the sequence box is locked. To edit the sequence, click to unlock. December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm Saving the Document Ordering Primers and Probes As a general guideline, select the primer/probe sets with a low Penalty score and a low amplicon length (if the Penalty score and Amplicon Length fields are not displayed, scroll to the right in the table). However, all primer/probe sets generated using default parameters meet primer and probe guidelines. For more information regarding Penalty scores, see Primer Express Software Version 3.0 Online Help. Before proceeding to other designs, be sure to save the Primer/Probe annotations and results found. Select File > Save As to save the document for future use. To order your selected primers and probes, refer to Chapter 4, Ordering Primers and Probes. IMPORTANT! Before running your samples, Applied Biosystems recommends that you run control samples to verify the performance of the selected primers and probes. 3 Primer Express Software 3.0 Getting Started Guide 33

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays Manually Designing Primers and Probes Manually Designing Primers and Probes You may choose to manually design primers and probes for a various reasons: Automated primer/probe design did not find primers or probes. To design primers and probes according to your own specifications. Creating an Allelic Discrimination Document Manually Designing the Allele 1 Probe Create an MGB allelic discrimination document and load a sequence file as you would for automatic primer/probe set design. See Creating an Allelic Discrimination Document on page 29. To design the probe for Allele 1: 1. In the Sequence tab, identify the SNP site and the putative probe sequence. 2. Assign the SNP target (see Assigning a SNP Target on page 30). 3. Select the sequence for the probe (13 to 25 bases) then select Edit > Copy with Allele 1. IMPORTANT! The Primer Probe Test Tool eliminates non-atcg bases. Before copying a sequence, change any non-atcg bases, or select a different region of the sequence. 4. Select Tools > Primer Probe Test Tool. 5. From the Document Type drop down menu, select the desired document type. Verify that the Parameter field is set to Default. For more information about parameters, see Primer Express Software Version 3.0 Online Help. 6. Paste (Ctrl+V) the annotated sequence in the Probe 1 field. The software displays the Tm, %GC, and the oligonucleotide length to the right of the Probe 1 field (see Figure 13. Primer Probe Test Tool dialog box on page 35). Note that the original Allele 1 variant base appears in lower case on the Primer Probe Test Tool. December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm 34 Primer Express Software 3.0 Getting Started Guide

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays Manually Designing Primers and Probes Figure 13. Primer Probe Test Tool dialog box 7. If the Tm is not between 65 C to 67 C, highlight a section of the sequence to view the corresponding Tm, %GC, and oligonucleotide length. Once the highlighted region results in the desired Tm, click on Trim to delete the non-highlighted bases. December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm Ensure the following guidelines are met (for more information on design guidelines, refer to Primer Express Software Version 3.0 Online Help): Amplicon Length 50 to 150 bases for optimum PCR efficiency. Probe Length 13 to 25 bases (13 to 30 bases if using conventional TaqMan probes). Tm 65 C to 67 C. % GC 30% to 80%. 5 end Cannot be a G residue. A G residue adjacent to the reporter dye will quench the reporter fluorescence somewhat, even after cleavage. Tm difference between probes Not greater than 1 C SNP site Locate in the middle third of sequence or toward 3 end but not in the last two bases of 3 end (see Figure 14. SNP site in an MGB probe on page 36). 3 Primer Express Software 3.0 Getting Started Guide 35

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays Manually Designing Primers and Probes SNP site If necessary, position the SNP site toward the 3 end. 5 3 N N N N N N N N N N N N N N N N N N N N N If possible, position the SNP site in the middle third of the probe. Do not position the SNP site in the last two bases. Figure 14. SNP site in an MGB probe Avoid the following motifs: Repeating oligonucleotides Avoid runs of identical nucleotides. If repeats are present, there must be fewer than four consecutive G residues. G residues on the 3 end Avoid 5 -...GGG-MGB-3 or 5 -...GGAG-MGB-3 Consecutive A residues Avoid six consecutive A residues anywhere in the probe. CC dinucleotides Avoid two or more CC dinucleotides in the middle of the probe, which can sometimes reduce signal. FAM -dye labeled probes If ordering FAM -dye labeled probes, avoid a G in the second position on the 5 end. For secondary structure design considerations, see Primer Express Software Version 3.0 Online Help. Note: If you cannot achieve the recommended Tm, or probe allele 1 is no longer within the guidelines, you can design using the complementary sequence. For more information, see Primer Express Software Version 3.0 Online Help. Manually Designing the Allele 2 Probe To design the probe for Allele 2: Note: Keep the Allele 1 and Allele 2 probe Tms within one degree of each other. 1. In the Sequence tab, select the sequence for the probe (13 to 25 bases and includes the SNP site) then select Edit > Copy with Allele 2. IMPORTANT! The Primer Probe Test Tool eliminates non-atcg bases. Before copying a sequence, change any non-atcg bases, or select a different region of the sequence. 2. Select Tools > Primer Probe Test Tool. December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm 36 Primer Express Software 3.0 Getting Started Guide

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays Manually Designing Primers and Probes 3. Paste (Ctrl+V) the sequence into the Probe 2 field. The Primer Probe Test Tool displays the Tm, %GC, and sequence length to the right of the field. Note that the original Allele 2 variant base will appear in lower case on the Primer Probe Test Tool. 4. If the Tm is not between 65 C to 67 C, highlight a section of the sequence to view the corresponding Tm, %GC, and oligonucleotide length of the highlighted region. Once the highlighted region results in the desired Tm, click on Trim to delete the non-highlighted bases. Keep in mind the general design guidelines previously listed on page 35. Manually Designing the Primers To design the Forward Primer: 1. Select a sequence (at least 25 bases) to the left of the probe. The sequence should be as close to the probe as possible without overlapping it. 2. Copy (Ctrl+C) the sequence. IMPORTANT! The Primer Probe Test Tool eliminates non-atcg bases. Before copying a sequence, change any non-atcg bases, or select a different region of the sequence. 3. On the Primer Probe Test Tool dialog box, paste (Ctrl+V) the sequence into the Fwd Primer field. The Primer Probe Test Tool displays the Tm, %GC, and the oligonucleotide length to the right of the Fwd Primer field. 4. If the Tm is not between 58 C to 60 C, highlight a section of the sequence to view the corresponding Tm, %GC, and oligonucleotide length as if those highlighted bases were deleted. Once the highlighted region results in the desired Tm, click on Trim to delete the non-highlighted bases. December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm Ensure the following guidelines are met (for more information on design guidelines, refer to Primer Express Software Version 3.0 Online Help): Amplicon Length 50 to 150 bases for optimum PCR efficiency. Optimal Primer Length 20 bases. Do not overlap primer and probe sequences. Tm 58 C to 60 C (Optimal Tm 59 C). % GC 30% to 80%. 3 end Make sure the last five nucleotides at the 3 end contain no more than two G + C residues. Avoid the following motifs: Repeating oligonucleotides Avoid runs of identical nucleotides. If repeats are present, there must be fewer than four consecutive G residues. For secondary structure design considerations, see Primer Express Software Version 3.0 Online Help. 3 Primer Express Software 3.0 Getting Started Guide 37

Chapter 3 Designing Primers and Probes for Allelic Discrimination Assays Manually Designing Primers and Probes To design the Reverse Primer: 1. In the sequence tab, select a sequence (at least 25 bases) to the right of the probe.the sequence should be as close to the probe without overlapping it. 2. Select Edit > Copy Complement. IMPORTANT! The Primer Probe Test Tool eliminates non-atcg bases. Before copying a sequence, change any non-atcg bases, or select a different region of the sequence. 3. On the Primer Probe Test Tool dialog box, paste (Ctrl+V) the sequence into the Rev Primer field. The Primer Probe Test Tool displays the Tm, %GC, and the oligonucleotide length to the right of the Fwd Primer field. 4. If the Tm is not between 58 C to 60 C, highlight a section of the sequence to view the corresponding Tm, %GC, and oligonucleotide length. Once the highlighted region results in the desired Tm, click on Trim to delete the non-highlighted bases. Be sure to keep the above guidelines in mind. Note that you can further customize your primer and probe set by editing the default parameter values found under the Parameters tab. For more information on editing parameters, see Primer Express 3.0 Software Online Help. Saving Primer and Probe Sequences Copy and paste the primer and probe sequences into a text document, then save for future reference. Ordering Primers and Probes To order primers and probes, see Chapter 4, Ordering Primers and Probes. IMPORTANT! Before running your samples, Applied Biosystems recommends that you run control samples to verify the performance of the selected primers and probes. December 16, 2004 3:54 pm, GSG_Chapter_4-03.fm 38 Primer Express Software 3.0 Getting Started Guide

Chapter 4 Ordering Primers and Probes Before You Begin See page 40 Designing Primers and Probes for Quantification Assays See page 40 See page 40 December 16, 2004 3:54 pm, GSG_Chapter_4-04.fm Designing Primers and Probes for Allelic Discrimination Assays Ordering Primers and Probes Overview Ordering the Selected Primers and Probes See page 40 See page 40 See page 40 4 Primer Express Software 3.0 Getting Started Guide 39

Chapter 4 Ordering Primers and Probes Overview Overview About This Chapter This chapter provides information on how to order your selected primer and probes. Ordering Primers and Probes After the Primer Express Software generates the table of candidate primers and probes, you can order those that best suit your needs. 1. In the Primer/Probe tab, select the primer and probe set you want to order. 2. Click on the Order tab. 3. Click on the toolbar to go the Applied Biosystems online store. 4. Log into the AB Store if you have an account, register if you are a new user. Ordering Primers 1. Below the ABI PRISM Primers/Probes heading, under the TaqMan Primers and Probes heading, click Sequence Detection Primers. 2. In the Product Information tab, select the check box next to the volume of primers to order. 3. Below the primer option you selected, click Customize. 4. Follow the instructions on the web page to specify any options. 5. Follow the instructions on the web page to enter or copy your sequence text. 6. Type the name for the Forward Primer, press Enter, then copy and paste your primer sequence from either the Order tab or the Primer Probe Test Tool (in the Primer Express software). 7. Type the name for the Reverse Primer, press Enter, then copy and paste your primer sequence from either the Order tab or the Primer Probe Test Tool (in the Primer Express software). 8. Click Continue. 9. Review your order, then click Add to Basket. If this completes your order (SYBR Green Dye assays), click Proceed to Checkout and follow the instructions on the web page to complete your order. Otherwise, click Continue Shopping to add Green Dye or probes to your order. December 16, 2004 3:54 pm, GSG_Chapter_4-04.fm 40 Primer Express Software 3.0 Getting Started Guide

Chapter 4 Ordering Primers and Probes Ordering Primers and Probes Ordering Probes 1. Above the Sequence Detection Primers heading, click the TaqMan Primers & Probes link. 2. Below the ABI PRISM Primers/Probes heading, click the TaqMan Primers & Probes link to expand the list. 3. Select TaqMan MGB Probes or TaqMan TAMRA dye Probes (if ordering conventional probes). 4. In the Product Information tab, select the check box next to the volume of probes to order. 5. Below the probe option you selected, click Customize. a. Follow the instructions on the web page to enter or copy your sequence text. If this probe is for allelic discrimination, be sure to specify the appropriate dyes. b. To order additional probes, follow the steps above. Otherwise, review your order, then click Add to Basket. c. Click Proceed to Checkout, then follow the instructions on the web page to complete your order. December 16, 2004 3:54 pm, GSG_Chapter_4-04.fm 4 Primer Express Software 3.0 Getting Started Guide 41

Chapter 4 Ordering Primers and Probes Ordering Primers and Probes December 16, 2004 3:54 pm, GSG_Chapter_4-04.fm 42 Primer Express Software 3.0 Getting Started Guide

Index A add DNA file 9, 19, 29 allele 2, 34, 36, 37 allelic discrimination creating the document 29 allelic discrimination assay defined 2 allelic discrimination assays assigning a SNP target 30 design guidelines 35 finding primers and probes 31 manually designing the allele 1 probe 34 manually designing the allele 2 probe 36 saving primer and probe sequences 38 saving the document 33 amplicon length 12, 14, 16, 17, 22, 24, 33, 35, 37 Annotating Sequences. See Online Help anti-sense strand 2, 3, 10, 30 anti-sense strand defined 2 Applied Biosystems contacting viii customer feedback on documentation viii Technical Communications viii Technical Support viii B Batch Process Tool. See Online Help D document defined 2 documentation feedback viii E Exporting. See Online Help F File format supported. See Online Help I installing Primer Express Software 4 O Online Help 6 Ordering TaqMan TAMRA dye Probes 41 ordering primers 40 ordering probes 41 Overview 8 P primer defined 3 Primer Express Software Version 2.0 4 Primer Express software version 2.0 5 primer length 17, 24, 37 Primer Probe Test Tool 13, 14, 15, 16, 17, 23, 24, 25, 34, 35, 36, 37, 38, 40 Printing. See Online Help probe defined 3 probe length 14, 35 Q quantification assay 3 Quantification Assays Manually Designing the Probe 13 Primer Design Guidelines 17, 37 quantification assays saving the document after automatic design 12 viewing results 11 quantification document 9 Quantification Workflow 8 R rapid assay design guidelines 2 registration code 6 S sense strand 9, 10, 19, 20, 29, 30 sense strand defined 3 SNP 2, 29, 30, 34, 35, 36 SNP site 29, 30, 34, 35, 36 SNP site in an MGB probe 36 Primer Express Software 3.0 Getting Started Guide 43

Index SNP Target 30 SNP target 30 SNP Target Tool 31 Starting and Exiting Primer Express Software 6 SYBR Green Dye Assays Primer Guidelines 24 SYBR Green Dye assays manually designing the forward primer 23 manually designing the primers 23 manually designing the reverse primer 25 saving primer sequences 25 viewing results 21 system requirements 3 T TaqMan Probe 3 TaqMan MGB Probe defined 3 TaqMan MGB Quantification 9 TaqMan Quantification 9 U Uninstalling Primer Express Software 4 44 Primer Express Software 3.0 Getting Started Guide

Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our Web site at www.appliedbiosystems.com. Applera is committed to providing the world s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: +1 650.638.5800 Toll Free (In North America): +1 800.345.5224 Fax: +1 650.638.5884 www.appliedbiosystems.com Part Number 4362460 Rev. B